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Resistance to current therapies still impacts a significant number of melanoma patients and can be regulated by epigenetic alterations. Analysis of global cytosine methylation in a cohort of primary melanomas revealed a pattern of early demethylation associated with overexpression of oncogenic transcripts. Loss of methylation and associated overexpression of the CSF 1 receptor (CSF1R) was seen in a majority of tumors and was driven by an alternative, endogenous viral promoter in a subset of samples. CSF1R was particularly elevated in melanomas with BRAF and other MAPK activating mutations. Furthermore, rebound ERK activation after BRAF inhibition was associated with RUNX1-mediated further upregulation of CSF-1R and its ligand IL-34. Importantly, increased CSF-1R and IL-34 overexpression were detected in an independent cohort of resistant melanomas. Inhibition of CSF-1R kinase or decreased CSF-1R expression by RNAi reduced 3-D growth and invasiveness of melanoma cells. Coinhibition of CSF-1R and BRAF resulted in synergistic efficacy in vivo. To our knowledge, our data unveil a previously unknown role for the autocrine-regulated CSF-1R in BRAF V600E resistance and provide a preclinical rationale for targeting this pathway in melanoma.

Bromodomain and extraterminal domain inhibitors (BETi) represent promising therapeutic agents for metastatic melanoma, yet their mechanism of action remains unclear. Here we interrogated the transcriptional effects of BETi and identified AMIGO2, a trans-membrane molecule, as a BET target gene essential for melanoma cell survival. AMIGO2 is upregulated in melanoma cells and tissues compared to human melanocytes and nevi, and AMIGO2 silencing in melanoma cells induces G1/S arrest followed by apoptosis. We identified the pseudokinase PTK7 as an AMIGO2 interactor whose function is regulated by AMIGO2. Epigenomic profiling and genome editing revealed that AMIGO2 is regulated by a melanoma-specific BRD2/4-bound promoter and super-enhancer configuration. Upon BETi treatment, BETs are evicted from these regulatory elements, resulting in AMIGO2 silencing and changes in PTK7 proteolytic processing. Collectively, this study uncovers mechanisms underlying the therapeutic effects of BETi in melanoma and reveals the AMIGO2-PTK7 axis as a targetable pathway for metastatic melanoma.

To the Editor ATRX is a member of the SWI/SNF family of chromatin remodelers, originally identified as mutated in patients with Alpha Thalassemia/Mental Retardation, X-linked syndrome. The protein product contains several highly conserved domains, including an ADD (ATRX-DNMT3-DNMT3L) domain that binds methylated histone H3 at lysine 9 and an ATPase domain responsible for its remodeling activities (Ratnakumar & Bernstein, 2013). Recently, whole genome sequencing studies identified ATRX mutations in multiple tumors, including those of neural crest cell origin: neuroblastoma, low-grade glioma and glioblastoma (Cheung et al., 2012; Heaphy et al., 2011a; Jiao et al., 2011; Kannan et al., 2012; Schwartzentruber et al., 2012). ATRX alterations encompass point mutations throughout the coding region as well as large N terminal deletions. While mechanistically unclear, ATRX mutations result in loss of protein as assessed by immunohistochemistry (IHC) and often correlate with alternative lengthening of telomeres (ALT) (Cheung et al., 2012; Heaphy et al., 2011a; Kannan et al., 2012; Schwartzentruber et al., 2012). To our knowledge, an investigation of ATRX in cutaneous melanoma is currently lacking. Our previous studies have demonstrated that decreased expression of histone variant macroH2A drives melanoma cell proliferation and metastasis (Kapoor et al., 2010), and that ATRX interacts with macroH2A to negatively regulate its association with chromatin (Ratnakumar et al., 2012). Taken together with recent reports of decreased ATRX protein in neural crest cell-derived tumors, we hypothesized that ATRX function might be compromised in melanoma. In order to test this hypothesis, we performed IHC on a panel of 23 benign nevi, 33 primary melanoma (≥1.0 mm deep) and 25 metastatic melanoma specimens that were formalin fixed paraffin embedded (FFPE) (Figure 1a, c). Slides were evaluated by two dermatopathologists in a blinded fashion using a scoring system based on number of positive nuclei and staining intensity (inter-rater correlation r=0.693, p

The histone variant mH2A is shown to be expressed at reduced levels in many melanomas. Loss of mH2A promotes tumour growth and metastasis, by transcriptional upregulation of CDK8, a known oncogene. This study therefore reveals a new tumour-suppression mechanism exerted by chromatin modification. The histone variant mH2A is shown to be expressed at reduced levels in many melanomas. Loss of mH2A promotes tumour growth and metastasis via transcriptional upregulation of CDK8, a known oncogene. This study therefore reveals a new tumour suppression mechanism exerted by epigenetic modifications. Cancer is a disease consisting of both genetic and epigenetic changes. Although increasing evidence demonstrates that tumour progression entails chromatin-mediated changes such as DNA methylation, the role of histone variants in cancer initiation and progression currently remains unclear. Histone variants replace conventional histones within the nucleosome and confer unique biological functions to chromatin1,2,3. Here we report that the histone variant macroH2A (mH2A) suppresses tumour progression of malignant melanoma. Loss of mH2A isoforms, histone variants generally associated with condensed chromatin and fine-tuning of developmental gene expression programs1,4,5,6, is positively correlated with increasing malignant phenotype of melanoma cells in culture and human tissue samples. Knockdown of mH2A isoforms in melanoma cells of low malignancy results in significantly increased proliferation and migration in vitro and growth and metastasis in vivo. Restored expression of mH2A isoforms rescues these malignant phenotypes in vitro and in vivo. We demonstrate that the tumour-promoting function of mH2A loss is mediated, at least in part, through direct transcriptional upregulation of CDK8. Suppression of CDK8, a colorectal cancer oncogene7,8, inhibits proliferation of melanoma cells, and knockdown of CDK8 in cells depleted of mH2A suppresses the proliferative advantage induced by mH2A loss. Moreover, a significant inverse correlation between mH2A and CDK8 expression levels exists in melanoma patient samples. Taken together, our results demonstrate that mH2A is a critical component of chromatin that suppresses the development of malignant melanoma, a highly intractable cutaneous neoplasm.

Epigenetic changes in cancer are thought to contribute to the regulation of invasion and metastasis. To study this at a genome-wide level in melanoma, we analyzed the methylome of 44 cases of malignant melanoma. We saw widespread demethylation occurring preferentially outside of CpG islands. Comparison of primary and metastatic lesions showed demethylation occurs early during carcinogenesis with few additional alterations in advanced tumors. The colony stimulating factor-1 receptor was aberrantly expressed and hypomethylated in nearly all cases. Its expression was validated by IHC and RNA-FISH on primary tumors and by qPCR, Western blotting and FACS in BRAF mutant and WT cell lines. CSF1R can be aberrantly expressed via an upstream LTR element in Hodgkin's lymphoma. After analyzing our patient samples and cell lines, we have found this aberrant transcript may be the dominant form in melanoma as well. Expression of one of its ligands IL34 was also shown in the cell lines by both ELISA and qPCR pointing to a potential autocrine regulatory loop. The effects of a small molecule inhibitor, PLX3397 as well as shRNA-mediated knockdown of the receptor were investigated in 2D and 3D cell culture. We saw inhibition of cell growth, smaller colony size, increased apoptosis and decreased invasiveness suggesting a functional role for CSF-1R in melanoma. Treatment of melanoma with BRAF-V600E inhibitors is effective for a time, but resistance invariably develops. The feedback activation of EGFR, BRAF amplification, BRAF splice variants and others are known to aid in the acquisition of resistance and the rebound activation of the MAPK-pathway. We are suggesting a role for CSF1R in this process. In Western experiments, the rebound of phospho-ERK after BRAF inhibitor treatment was accelerated with the addition of CSF1R ligands, or delayed with PLX3397, also attenuating AKT phosphorylation. Melanoma cells stably expressing shRNA against CSF1R recapitulated the effects of the inhibitor. Assaying the cells at different time points during a long-term V600E inhibitory experiment, we saw increasing levels of the transcription factor RUNX1, followed by increasing levels of IL34 and of the receptor, as well as its maturation, and presentation on the cell surface. shRNA-mediated knockdown of RUNX1 resulted in lower levels of the CSF1R and IL34 transcripts and delayed the rebound. Analysis of primary RNA-Seq data showed an increase in RUNX1, CSF1R and IL34 expression in resistant tumors. Co-inhibition of CSF1R and BRAF was also tested and resulted in synergistic blockade of cell growth in vitro and xenograft growth in vivo.The CSF1R inhibitor, PLX3397 is currently in clinical trials for glioblastoma, prostate, breast cancers and other cancers. These data present a preclinical rationale for its study in malignant melanoma. Citation Format: Orsolya Giricz, Yongkai Mo, Kimberly B. Dahlman, Xiomaris M. Cotto-Rios, Chiara Vardabasso, Hoa Nguyen, Bernice Matusow, Matthias Bartenstein, Veronika Polishchuck-Lee, Douglas B. Johnson, Tushar B. Bhagat, Rafe Shellooe, Elizabeth Burton, Gaston Habets, John M. Greally, Yiting Yu, Gideon Bollag, Paraic A. Kenny, Kith Pradhan, E. Richard Stanley, Emily Bernstein, Evripidis Gavathiotis, Brian L. West, Jeffrey A. Sosman, Amit Verma. Aberrant expression of CSF1R in melanoma is driven through an endogenous viral promoter and it contributes to malignant growth and BRAF-inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2515.

Histone variants are emerging as key regulatory molecules in cancer. We report a unique role for the H2A.Z isoform H2A.Z.2 as a driver of malignant melanoma. H2A.Z.2 is highly expressed in metastatic melanoma, correlates with decreased patient survival, and is required for cellular proliferation. Our integrated genomic analyses reveal that H2A.Z.2 controls the transcriptional output of E2F target genes in melanoma cells. These genes are highly expressed and display a distinct signature of H2A.Z occupancy. We identify BRD2 as an H2A.Z-interacting protein, levels of which are also elevated in melanoma. We further demonstrate that H2A.Z.2-regulated genes are bound by BRD2 and E2F1 in an H2A.Z.2-dependent manner. Importantly, H2A.Z.2 deficiency sensitizes melanoma cells to chemotherapy and targeted therapies. Collectively, our findings implicate H2A.Z.2 as a mediator of cell proliferation and drug sensitivity in malignant melanoma, holding translational potential for novel therapeutic strategies.

Histone variants are attracting attention in the field of cancer epigenetics. Our study has established a novel role for the uncharacterized histone variant H2A.Z.2 as a driver of malignant melanoma. H2A.Z.2 promotes cellular proliferation by recruiting BRD2 and E2F1 to E2F target genes and facilitating their transcription. High H2A.Z.2 expression correlates with poor survival in patients, and its depletion sensitizes cells to chemotherapy and targeted therapies.

Malignant melanoma is the most lethal form of skin cancer with rising incidence. Once metastasis occurs, patients have a dismal prognosis, largely due to limited systemic treatment with chemotherapy and resistance to targeted therapies. Thus, effective therapies with long-term responses are currently lacking. Although much effort has focused on characterizing and targeting the genetic alterations in melanoma, the identification of epigenetic players remains poorly understood. Chromatin dynamics have recently been shown to exert a critical function in a number of cancers, including melanoma, and emerging evidence points towards a role of histone variants as key regulatory molecules in cancer. H2A.Z is a highly conserved H2A variant, harboring two different isoforms in vertebrates, H2A.Z.1 and H2A.Z.2. High levels of H2A.Z promote cell proliferation in breast, prostate and bladder cancers, however studies so far have focused primarily on H2A.Z.1 or did not clearly distinguish between the two isoforms. Here, we report a role for the unappreciated isoform H2A.Z.2 as a mediator of cell proliferation and drug sensitivity in malignant melanoma. To our knowledge, this is the first evidence to implicate a distinct role for this H2A.Z isoform in any tumor type. While both H2A.Z.1 and H2A.Z.2 are highly expressed in metastatic melanoma and correlate with decreased patient survival, only H2A.Z.2 deficiency results in impaired cellular proliferation, which occurs through a G1 to S arrest. Integrated gene expression and ChIP-seq analyses revealed that H2A.Z.2 positively regulates E2F target genes, which are highly expressed and acquire a distinct H2A.Z occupancy signature over the promoter and gene body in metastatic cells. We further identified the BET (bromodomain and extraterminal domain) family member BRD2 as an H2A.Z-interacting protein in melanoma cells, and our data suggest that H2A.Z.2 exerts its oncogenic function by maintaining the global levels of BRD2 and histone H4 acetylation. Furthermore, H2A.Z.2 depletion sensitizes melanoma cells to targeted therapies and chemotherapy. Collectively, our findings implicate H2A.Z.2 as a driver of melanoma pathogenesis. Owing to the fact that histone modification is a reversible process, H2A.Z.2 and BRD2 hold translational potential for novel therapeutic strategies. Citation Format: Chiara Vardabasso, Alexandre Gaspar-Maia, Sebastian Punzeler, David Valle-Garcia, Dan Hasson, Tobias Straub, Eva C. Keilhauer, Thomas Strub, Taniya Panda, Miguel F. Segura, Chi-Yeh Chung, Amit K. Verma, Matthias Mann, Eva Hernando, Sandra B. Hake, Emily Bernstein. Histone variant H2A.Z.2 mediates proliferation and drug sensitivity of malignant melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A12.